Equilibrium dialysis is a procedure for measuring the concentration of free, relatively small molecules in a sample. The procedure originally was designed to study the quantitative aspects of immunity reactions, and in particular to study the combination of antibodies with simple haptens. See J. Marrack and F. C. Smith, Brit. J. Exptl. Path., 13, 394 (1932). Over the years, the procedure has been employed primarily in immunological studies; see, e.g., F. Haurowitz and F. Breinl, Z. Physiol. Chem., 214, 111 (1933); H. N. Eisen and F. Karush, J. Am. Chem. Soc., 71, 363 (1949); and D. N. Weir, Editor, "Handbook of Experimental Immunology," Second Edition, Blackwell Scientific Publications, Oxford, 1973, pages 16.1-16.21.
In principle, equilibrium dialysis can be employed to determine the concentration of any relatively small molecule in a given sample; the only requirement is that the material to be measured must pass freely through a semi-permeable membrane. Once equilibrium has been achieved, it is only necessary to analyze the dialysate for the desired material.
As a practical matter, equilibrium dialysis appears to be directed primarily to the determination of the concentration of free or unbound haptens which are present in blood serum or plasma samples. Thus, the discussion herein will be directed toward that end-use, although it should be apparent that the invention disclosed herein is not to be limited by such discussion. Furthermore, for simplicity the discussion will be limited to the assay of free thyroxine (T.sub.4) in blood serum or plasma.
It should be noted that the need for equilibrium dialysis in such a determination arises because a large proportion of the hapten is bound to much larger molecules, such as serum proteins, which do not pass through a semi-permeable membrane. The free or unbound portion of the hapten is the physiologically important quantity, which renders such quantity clinically important, as well.
At the present time, the only generally-accepted reference method for measuring the concentration of free thyroxine in blood serum or plasma samples is by equilibrium dialysis. Basically, the procedure involves placing the serum sample in a dialysis bag along with radiolabeled thyroxine. The bag is placed in a known volume of liquid and typically incubated for 16 hours. At the end of the incubation period, a sample of the dialysate is taken and the radioactivity of such sample is measured. In practice, however, it also is necessary to go through a procedure to remove radioactive breakdown products from the dialysate. Such procedure involves adding to the dialysate pooled human serum to absorb free thyroxine. A resin then is added to absorb the radioactive breakdown products, which resin subsequently is removed from the dialysate. Unfortunately, the resin addition step is extremely critical. If the resin is in the dialysate for too short a time, it will not pick up all of the breakdown products, thereby leading to erroneously high results. Conversely, if the resin is in contact with the dialysate for too long a time, it also strips free thryoxine from the added serum protein, leading to erroneously low results. Thus, it is necessary to critically control the time and conditions of the resin addition and removal steps in order to obtain correct results.